KR101808196B1 - New small molecules from scolopendra subspinipes mutilans and antithrombotic use thereof - Google Patents

Abstract

The invention wangjine (Scolopendra anti-platelet and anti-adherent pharmaceutical composition comprising as an active ingredient a compound represented by any one of formulas (1), (2) and (3) or a pharmaceutically acceptable salt thereof isolated from subspinipes The compounds represented by Chemical Formulas 1, 2 and 3 are activated partial thromboplastin time (aPTT) and prothrombin time in human umbilical vein endothelial cells (HUVECs) prothrombin time, PT), inhibiting thrombin and activated factor X (FXa), inhibiting thrombin-catalyzed fibrin polymerization and platelet aggregation, inhibiting thrombopoietic model and antithrombotic effect in arterial thrombosis model Inhibits protein kinase C (PKC) activity and calcium mobilization, and inhibits the anticoagulant effect in mice , And by reducing the ratio of plasminogen activator inhibitor-1 (PAI-1) to tissue plasminogen activator (t-PA) Platelet agents and anticoagulants.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to novel compounds derived from Wang Jiin,

The present invention relates to novel compounds isolated from Scolopendra subspinipes mutilans L. Koch and their anti-thrombotic uses, and more particularly to novel compounds isolated from Wang Ji Nong , acylated polyamines and sulfated quinoline alkaloids, Acylated polyamines, sulfated quinoline alkaloids and indoleacetic acid.

Thrombus formation is a critical event in the pathophysiology of atherosclerotic cardiovascular diseases (CVD) (Non-Patent Document 1). Thrombotic induced myocardial infarction or ischemic stroke is a major cause of CVD-related deaths. Thrombus formation due to an abnormal clotting process is often observed in the arteries or veins and may result in reduced blood flow or ischemia (Non-Patent Document 1). Thrombus formation is associated with blood cell wall damage, water-soluble cell adhesion, coagulation, and fibrinolysis (Non-Patent Documents 1 and 2). A global leading cause of death is cardiovascular disease and consequently thrombosis (non-patent document 1). Platelet activity in atherosclerotic arteries is important for the development of arterial thrombosis, and therefore precise control of platelet function is essential for preventing thrombotic events (Non-Patent Document 3). Most thrombotic procedures require anticoagulant therapy. This explains the current efforts to develop specific and potential anticoagulants and antithrombotic agents. Studies on new physiologically active compounds and drugs with other mechanisms of action, increased efficacy and lower toxicity are highly needed (Non-Patent Document 1).

The term " Scolopendra subspinipes mutilans L. Koch" is used to refer to stroke-related unilateral paralysis, epileptic seizures, tetanus, and pain, hereinafter abbreviated as "SSM" Which is a medicinal resource listed in the herbal medicine and Chinese pharmacopoeia (Non-Patent Documents 4 and 5). The traditional application of SSM in unilateral paralysis associated with stroke and stroke has attracted interest in discovering anticoagulants from zines. Up to now, it has been demonstrated that peptides and proteins in SSM poisons have antithrombotic effects (Non-Patent Documents 6 to 9). In addition, studies based on evidence for SSM have led to the discovery of potential peptides to be developed as effective analgesics as morphine (Non-Patent Document 10). Quinoline alkaloids are specified as representative small molecule metabolites of SSM, and there are some chemical studies on secondary metabolites of SSM (Non-Patent Documents 11-14).

As a commercial anticoagulant, heparin has been used for prevention of thromboembolic diseases of veins for more than 60 years (Non-Patent Documents 15 and 16). However, heparin has been shown to be effective in inhibiting thrombin bound to fibrin, inefficiency in congenital or acquired antithrombin deficiency, development of thrombocytopenia, increased risk of thromboembolic disease if the therapeutic response is not achieved, (Such as increased risk of bleeding) (Non-Patent Documents 16 and 17). Also, the amount of available heparin is small in the small lung or porcine small intestine where heparin is mainly extracted (Non-Patent Document 17). Therefore, the need to find alternative sources of anticoagulants has increased with the demand for safe anticoagulant therapy.

As a result of efforts to discover a small molecule anticoagulant from the entire substance of clinically used Wangjing (SSM), the present inventors have found that a novel acylated polyamine (Compound 1) and a sulfated quinoline alkaloid 2), and five known alkaloids, 8-hydroxy-4-quinolone (Compound 3) and indole acetic acid (Compound 4) Inhibits the production of thrombin and activated factor X (FXa), and prothrombin time (PT), activated partial thromboplastin time (aPTT) and fibrinolytic activity ), Thereby completing the present invention.

Fares A. Winter cardiovascular diseases phenomenon. N Am J Med Sci 2013, 5 (4): 266-279. Weitz JI, Hirsh J, Samama MM. New anticoagulant drugs: the Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy. Chest 2004, 126 (3 Suppl): 265S-286S. Davi G, Patrono C. Platelet activation and atherothrombosis. N Engl J Med 2007, 357 (24): 2482-2494. Cai MD, Choi S-M, Song BK, Son I, Kim S, Yang EJ. Scolopendra subspinipes mutilans attenuates neuroinflammation in symptomatic hSOD1G93A mice. J Neuroinflammation 2013, 10: 131 / 131-131 / 139, 139 pp. Ma W, Zhang D, Zheng L, Zhan Y, Zhang Y. Potential roles of Centipede Scolopendra extracts as a strategy against EGFR-dependent cancers. Am J < / RTI > Translated 2015, 7 (1): 39-52. Kong Y, Li S, Shao Y, He Z-I, Chen M-m, Ming X, et al. Antithrombotic Peptides from Scolopendra subspinipes mutilans Hydrolysates. International Journal of Peptide Research and Therapeutics 2014, 20 (2): 245-252. Kong Y, Huang SL, Shao Y, Li S, Wei JF. Purification and characterization of a novel antithrombotic peptide from Scolopendra subspinipes mutilans. Ethnopharmacol 2013, 145 (1): 182-186. Kong Y, Shao Y, Chen H, Ming X, Wang JB, Li ZY, et al. A Novel Factor Xa-Inhibiting Peptide from Centipedes Venom. Int J Pept Res Ther 2013, 19: 303-311. Liu ZC, Zhang R, Zhao F, Chen ZM, Liu HW, Wang YJ, et al. Venomic and transcriptomic analysis of centipede Scolopendra subspinipes dehaani. J Proteome Res 2012, 11 (12): 6197-6212. Yang S, Xiao Y, Kang D, Liu J, Li Y, Undheim EA, et al. Discovery of a selective NaV1.7 inhibitor from centipede venom with analgesic efficacy exceeding morphine in rodent pain models. Proc Natl Acad Sci U SA 201, 110 (43): 17534-17539. Moon SS, Cho NS, Shin J, Seo Y, Lee CO, Choi SU. Jineol, a cytotoxic alkaloid from the centipede Scolopendra subspinipes. Journal of Natural Products 1996,59 (8): 777-779. Fu YD, Li ZL, Pu SB, Qian SH. Two new compounds from Scolopendra multidens Newport. J Asian Nat Prod Res 2013, 15 (4): 363-367. Yoon MA, Jeong TS, Park DS, Xu MZ, Oh HW, Song KB, et al. Antioxidant effects of quinoline alkaloids and 2,4-di-tert-butylphenol isolated from Scolopendra subspinipes. Biological & Pharmaceutical Bulletin 2006, 29 (4): 735-739. Noda N, Yashiki Y, Nakatani T, Miyahara K, Du XM. A novel quinoline alkaloid possessing a 7-benzyl group from the centipede, Scolopendra subspinipes. CHEMICAL & PHARMACEUTICAL BULLETIN 2001, 49 (7): 930-931. Wells PS, Forgie MA, Rodger MA. Treatment of venous thromboembolism. JAMA 2014, 311 (7): 717-728. Hirsh J, Raschke R, Warkentin TE, Dalen JE, Deykin D, Poller L. Heparin: mechanism of action, pharmacokinetics, dosing considerations, monitoring, efficacy, and safety. Chest 1995, 108 (4 Suppl): 258S-275S. Pereira MS, Melo FR, Mourao PA. Is there a correlation between structure and anticoagulant action of sulfated galactans and sulfated fucans Glycobiology 2002, 12 (10): 573-580.

The object of the present invention is to provide novel acylated polyamines (Compound 1) and sulfated quinoline alkaloids (Compound 2) isolated from Scolopendra subspinipes mutilans L. Koch.

Still another object of the present invention is to provide an anticoagulant which exhibits anticoagulant activity and anti-platelet activity, such as an acylated polyamine (Compound 1), a sulfated quinoline alkaloid (Compound 2), and indole acetic acid (Compound 4) And to provide a usable use.

In order to achieve the above object, the present invention provides a compound represented by the following formula (1) or (2)

[Chemical Formula 1]

; 및

; And

(2)

.

.

The present invention also provides a composition comprising the compound represented by the above formula (1) or (2).

The present invention also provides an antithrombotic, anti-platelet or anti-adherent pharmaceutical composition comprising, as an active ingredient, the compound represented by Formula 1, Formula 2, or Formula 3:

(3)

.

.

The present invention also provides an antithrombotic, anti-platelet or anti-hypertensive health functional food containing the compound represented by the above formula (1), (2) or (3) as an active ingredient.

The present invention also provides a pharmaceutical composition for the prevention and treatment of cardiovascular diseases, which comprises the compound represented by the above formula (1), (2) or (3) as an active ingredient.

In addition, the present invention provides a health functional food for prevention and improvement of cardiovascular diseases containing the compound represented by Chemical Formulas 1, 2, or 3 as an active ingredient.

Compounds 1, 2 and 4 according to the present invention inhibit the exogenous and endogenous blood coagulation pathways through inhibition of FXa and thrombin production in HUVEC, inhibit TNF-a-induced secretion of PAI-1, By inhibiting platelet aggregation inside and outside and specifically preventing protein kinase C and calcium mobilization, these compounds can be usefully used as an effective ingredient for the treatment or prevention of vascular diseases associated with coagulation.

Fig. 1 shows a schematic view of a Scolopendra subspinipes mutilans L. Koch).
FIG. 2 is a diagram showing the HMBC correlation (?) And the COZY correlation (?) Of the compounds 1 and 2.
Figure 3 is a graph showing the effect of fibrin polymerization, platelet aggregation and their cytotoxicity of compounds 1-4 (compound 1: white box; compound 2: light gray box; compound 3: dark gray box; Color box, where D used 0.2% DMSO as vehicle control):
Figure 3 A is a graph showing the degree of fibrin polymerization catalyzed by thrombin.
Figure 3B is a graph showing the degree of human platelet aggregation induced by 2 [mu] M U46619 in vitro.
FIG. 3C is a graph showing the results of in vitro monitoring of the degree of mouse platelet aggregation induced by 2 μM U46619. FIG.
FIG. 3D is a graph showing cell viability using MTT assay.
Figure 4 is a graph showing the effect of compounds 1-4 on carotid thrombosis and acute thrombosis model (compound 1: white box; compound 2: light gray box; compound 3: dark gray box; and compound 4: black box; Tiro: a positive control using tyrofiban, where D used 0.2% DMSO as vehicle control):
Figure 4A is a graph showing the time for large thrombus formation.
FIG. 4B is a graph showing the size score of the thrombus 60 minutes after FeCl ₃ treatment.
FIG. 4C shows the results of the administration of each compound intravenously, followed by administration of a mixture of collagen (C, 500 μg / kg) and epinephrine (E, 50 μg / kg) It is a graph showing the change of thrombus.
Figure 5 shows the effect of compound 1-4 on PKC activity and intracellular calcium mobilization (compound 1: white box; compound 2: light gray box; compound 3: dark gray box; and compound 4: black box ; Tiro: a positive control using tyrofiban, where D used 0.2% DMSO as vehicle control):
FIG. 5A is a graph showing the results of measurement of MARCKS phosphorylation using Western blotting. FIG.
FIG. 5B is a graph showing the degree of [Ca ^{2 +} ] _i increase by addition of U46619 in human platelets loaded with Fura-2.
FIG. 5C is a graph showing the degree of [Ca ^{2 +} ] _i increase by thrombin addition in human platelets loaded with Fura-2.
Figure 6 is a graph showing the inactivation and production of thrombin and factor Xa of compounds 1-4 (compound 1: white box; compound 2: light gray box; compound 3: dark gray box; and compound 4: black Box A or B: Argatroban or Libaroxan as a positive control, where D used 0.2% DMSO as vehicle control):
FIG. 6A is a graph showing the degree of inhibition of thrombin (Th) using a chromogenic assay.
FIG. 6B is a graph showing the suppression degree of the factor Xa (FXa) using color development analysis.
6C is a graph showing the degree of production of thrombin by HUVEC.
FIG. 6D is a graph showing the degree of production of the factor Xa (FXa) by the HUVEC.
Figure 7 is a graph showing the effect of compound 1-4 on PAI-1 and tPA secretion (compound 1: white box; compound 2: light gray box; compound 3: dark gray box; and compound 4: black box; Where D used 0.2% DMSO as vehicle control): < RTI ID = 0.0 >
FIG. 7A is a graph showing the PAI-1 concentration measured in HUVEC culture fluid cultured in the presence or absence of TNF-α. FIG.
Fig. 7B is a graph showing the tPA concentration measured in HUVEC cultures cultured in the presence or absence of TNF-a.
Figure 7C is a graph showing PAI-1 / t-PA ratios in HUVECs activated with TNF-α.
Figure 8 is a picture showing the mechanism of the antithrombotic activity of the compounds 1, 2 and 4.

Hereinafter, the present invention will be described in detail.

The present invention provides a compound represented by the following formula (1) or (2):

[Chemical Formula 1]

; 및

; And

(2)

.

.

The compound represented by the general formula (1) or (2) of the present invention can be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid and the like, aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, Derived from organic acids such as acetic acid, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid and the like. Examples of such pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, But are not limited to, but are not limited to, but are not limited to, but are not limited to, but are not limited to, halides, halides, halides, halides, halides, halides, But are not limited to, lactose, sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, Methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene sulfonate, chlorobenzene Sulfonates, methanesulfonates, propanesulfonates, naphthalene-1-sulfonates, and the like, as well as sulfonates such as benzyl sulfonate, sulfonate, xylene sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, -Sulfonate, naphthalene-2-sulfonate, mandelate, and the like.

The acid addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving a derivative of the formula (1) in an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile and the like, Followed by filtration and drying. Alternatively, the solvent and excess acid may be distilled off under reduced pressure, followed by drying and crystallization in an organic solvent.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or an alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. In addition, the corresponding salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable salt (such as silver nitrate).

Furthermore, the present invention encompasses not only the compound represented by the above formula (1) or (2) and pharmaceutically acceptable salts thereof, but also solvates, hydrates and the like which can be prepared therefrom.

The novel compounds of the present invention are compounds isolated from Scolopendra subspinipes mutilans L. Koch and are characterized in that they have a chemical structure which is not known before.

In addition,

A step of extracting Scolopendra subspinipes mutilans L. Koch with ethanol to obtain an ethanol extract (step 1);

The ethanol extract obtained in step 1 is suspended in water and then fractionated successively using ethyl acetate (EtOAc) and n-butanol (BuOH) to obtain an n-butanol fraction (step 2); And

And separating and purifying the n-butanol fraction obtained in the step 2 (step 3).

Step 2 above may comprise directly separating the compound using high performance liquid column chromatography (prep HPLC) with ethyl acetate (EtOAc) or n-butanol (BuOH) fractions.

The present invention also provides a composition comprising the compound represented by the above formula (1) or (2).

The composition may be prepared in a form comprising the compounds according to the invention and comprising a carrier or carrier suitable for use in pharmaceutical or food preparations.

The present invention also provides an antithrombotic, anti-platelet or anti-adherent pharmaceutical composition comprising as an active ingredient a compound represented by the following general formula (1), (2) or (3)

[Chemical Formula 1]

;

;

(2)

; 및

; And

(3)

.

.

The compounds of formula (I), (II) and (III) of the present invention prolong aPTT and PT, inhibit thrombin and FXa in human umbilical vein endothelial cells (HUVECs), inhibit thrombin-mediated fibrin polymerization and platelet aggregation Inhibit PKC activity and calcium mobilization in cells and decrease the ratio of PAI-1 to t-PA, thereby increasing the anti-thrombotic effect in the pulmonary embolism model and the arterial thrombosis model mouse, And an effective component of an anticoagulant.

When the composition according to the present invention is formulated, it may be prepared using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used.

Solid formulations for oral administration include tablets, pills, powders, granules, capsules, troches, and the like, which may contain one or more excipients such as starch, calcium carbonate, Sucrose, lactose, gelatin or the like. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions or syrups. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances and preservatives may be included. have.

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Examples of the non-aqueous solvent and suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 81, cacao butter, taurine, glycerol, gelatin and the like.

The composition according to the present invention may be administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will depend on the type of disease, severity, , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the compound according to the present invention may vary depending on the age, sex, and body weight of the patient. Generally, 0.001 mg to 1000 mg, preferably 0.01 mg to 100 mg, per kg body weight is administered daily or every other day Or one to three times a day. However, the dosage may be varied depending on the route of administration, the severity of the disease, sex, weight, age, and the like, and therefore the dose is not limited to the scope of the present invention by any means.

The present invention also provides an antithrombotic, anti-platelet or anti-hypertensive health functional food containing the compound represented by the above formula (1), (2) or (3) as an active ingredient.

Here, the term "health functional food" is produced by using raw materials or ingredients (functional raw materials) having functions useful for nutrients and human body which are likely to be deficient in daily eating, and is intended to maintain the normal function of the human body, But is not limited to, and is not meant to exclude health food in the usual sense.

The compounds of the present invention can be added directly to food or used together with other food or food ingredients, and can be suitably used according to conventional methods. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (for prevention or improvement). In general, the amount of the compound in the health functional food may be 0.01 to 90 parts by weight based on the total weight of the food. However, when consumed for a long period of time for the purpose of health and hygiene or for health control purposes, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

The health functional food of the present invention has no particular limitation on other ingredients other than those containing the above-mentioned compounds. Specifically, various flavoring agents such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and thickening agents (cheese, chocolate etc.), pectic acid and its salts, alginic acid and its salts, Protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. These components may be used independently or in combination. The ratio of such additives is not so critical, but is generally selected in the range of 0.1 to about 20 parts by weight per 100 parts by weight of the compound of the present invention.

The present invention also provides a pharmaceutical composition for the prevention and treatment of cardiovascular diseases, which comprises the compound represented by the above formula (1), (2) or (3) as an active ingredient.

In addition, the present invention provides a health functional food for prevention and improvement of cardiovascular diseases containing the compound represented by Chemical Formulas 1, 2, or 3 as an active ingredient.

The cardiovascular disease is preferably myocardial infarction or ischemic stroke caused by blood coagulation.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples.

However, it should be understood that the present invention is not limited to the embodiments and examples described below, but may be embodied in various other forms. The following embodiments and examples are intended to be illustrative, It is provided to fully inform the owner of the scope of the invention.

Example 1 Extraction and Separation of Small Molecule Alkaloid from SSM

The dried SSM (521 g) (purchased from the Jungsan herb medicine market, Korea) was extracted with ethanol (5 L × 4) at room temperature, and the obtained extract was concentrated under vacuum to obtain a brown ethanol extract (110.0 g). The brown ethanol extract was suspended in water and then sequentially fractionated using ethyl acetate (EtOAc) and n-butanol (BuOH) to obtain EtOAc water soluble fraction (SS-1, 60.0 g) and BuOH water soluble fraction (SS- ) And a residue (SS-3, 40.0 g).

The EtOAc water soluble fraction (SS-1) was eluted with hexane / EtOAc 40: 1, 20: 1, 10: 1, and 4: 1; Hexane / EtOAc / MeOH 2/1 / 0.2; CHCl ₃ / MeOH 6: 1; (SS-1a-h) by washing with MeOH as eluent after applying to vacuum-liquid chromatography (VLC) using CHCl ₃ / MeOH / H ₂ O 3: ≪ / RTI > At this time, VLC was performed on Merck silica gel (70-230 mesh). Fraction SS-1d (8.0 g) eluted with hexane / EtOAc 4: 1 was fractionated with hexane and MeOH to give jineol (60.0 mg). Fraction SS-1f (1.6 g) eluted with CHCl ₃ / MeOH 6: 1 was applied to prep HPLC using a gradient of MeOH to 10-100% water to give indole acetic acid (2- (1 H -indol-3yl ) acetic acid) (t _R 38.0 bun, 1 mg), 3- methoxy-8-hydroxy-quinolin-2-one (3-methoxy-8-hydroxy -quinolin-2-one) (t R, 42.0 bun , 22.0 mg) and 3,4-dimethoxy-8-hydroxy-quinolin-2 source (3,4-dimethoxy-8-hydroxy -quinolin-2-one) (t R, 52.0 bun, 36.0 mg) of Respectively. Fraction SS-1g (700 mg) eluted with CHCl ₃ / MeOH / H ₂ O 3: 1: 0.1 was isolated using preparative reverse phase HPLC (prep HPLC) using a gradient of MeOH to 15-85% It was separated by an 8-hydroxy-4-quinolone (8-hydroxy-4-quinolone ) (t R, 37.0 bun, 3.6 mg). The prep HPLC separation was performed on a YMC C18 column (250 × 20.0 mm, 5 μm) or a Kinetex Biphenyl column (250 × 21.5 mm, 5 μm) at a flow rate of 5 mL / min.

The BuOH water soluble fraction (SS-2, 7.4 g) was eluted with hexane / EtOAc / MeOH 2/1 / 0.2; _{CHCl 3 / MeOH 8: 1,} 6: 1, and 4: 1; After applying to VLC using CHCl ₃ / MeOH / H ₂ O 2: 1: 0.1, mobile phase was washed with 100% MeOH to obtain six fractions (SS-2a-f). The combined fractions SS-2d and SS-2e (eluted with CHCl ₃ / MeOH 6: 1 and 4: 1, 7.5 g) were applied to medium-pressure liquid chromatography (MPLC) -2e-I to SS-2e-VIII). At this time, the MPLC was performed using a Biotage Isolera apparatus equipped with a reverse phase C18 SNAP Cartridge KPC18-HS (340 g, Biotage AB, Uppsala, Sweden). Fraction SS-2e-III (213 mg) was isolated by prep HPLC using isocratic solution conditions of 30% MeOH. The HPLC fractions eluted between 10 to 12.5 min using the prep HPLC with a mobile phase of 10% MeOH in water and purified by removing the coupling compound 2 (t _R, 32.0 bun, 15.0 mg). Fraction SS-2e-V (89 mg) was applied to prep HPLC using a gradient of MeOH-H ₂ O (20% methanol, 25 min; 20-25%, 30 min; It was separated by a compound 1 (t _R, 41.0 bun, 1.0 mg). Fraction SS-2e-VI (85 mg ) to _{MeOH / H 2 O (1:} 1) (scolopendrine) (t R, 12.4 bun gave pen to Driscoll and purified by prep HPLC using an isocratic solution, 10.0 mg ).

Example 2 Identification and Structural Analysis of Small Molecule Alkaloids Isolated from SSM

The structure of the separated compounds was determined by MS, 1D, and 2D NMR analysis. NMR experiments were performed using a Bruker Avance III (600 MHz) spectrophotometer. IR data were recorded on thermoelectron US / Nicolet 380 (Madison, Wis., USA). HRESIMS data were acquired on a JMS 700 high resolution mass spectrometer (JEOL, Tokyo, Japan).

Specifically, Compound 1 was isolated as a yellow amorphous powder. The molecular formula of C ₁₂ H ₁₈ N ₄ O ₆ S was deduced from the pseudomolecular ion m / z 369.0841 (calculated [M + Na] ⁺ , m / z 369.0845) observed in the HR-ESI-MS data. ^{The 1} H and ¹³ C NMR spectra showed distinct resonances for the agartin moiety (Table 1). The NMR spectra closely corresponded to those of gentisic acid (GA). Comparing the proton chemical shifts of gentisic acid, the protons of H-2 and H-4 in compound 1 were significantly reduced by the downfield (delta 1-delta GA 0.35 and 0.31, respectively), indicating the presence of sulfate functionality at C-3 of compound 1 . A strong HMBC correlation from benzene protons of δ _H 6.85 (H-5) to quaternary carbons of δ _C 145.7 and up-fielded carbon chemical shift of about 5 ppm meant that the sulfate was replaced at C-3. The HMBC correlation between the carbonyl carbon at H2-1 'and delta _C 170.8 (C-7) at delta _H 3.44 allows the binding of agmatine and gentisic acid fragments and the binding of gentisic acid agmatine amide-3-sulfate). Compound 1 can be categorized as acylated polyamines which are reported to be derived from the poison of the arachnids (McCormick KD, et al., J Chem Ecol 1993, 19 (10): 2411-2451). Except for the polyamine chain, the structure of Compound 1 is associated with a toxin isolated from the venom of the spider Tegenaria agrestis (Schroeder FC, et al., Proc Natl Acad Sci 2008, 105 (38): 14283-14287). Compared to the structure of the known acylated polyamines, Compound 1 exhibits a unique chemical form for classification.

Compound 1: yellow amorphous powder; UV (MeOH)? Max (log?) 240 sh (4.22), 308 (3.61) nm; IR (KBr)? Max 3357, 1657, 1547, cm ^-1 ; ¹ H and ¹³ C NMR, see Table 1; HR-ESI-MS m / z 369.0841 [M + Na] ⁺ (calculated for C ₁₂ H ₁₈ N ₄ O ₆ SNa, m / z 369.0845).

Compound 2 was isolated as a yellow amorphous powder and showed a unique NMR signal similar to that of jineol (Moon SS, et al., J Nat Prod 1996, 59 (8): 777-779) (Table 2). The molecular formula C ₉ H ₇ NO ₅ S (calculated [M + Na] ⁺ , m / z 263.9943) was determined from the molecular ion peak m / z 263.9935 of the HR-ESI-MS data, Derivative. The chemical shift (δ _C 149.0 (C-8)) of the carbon which was approximately 5 ppm over zeinol indicated the presence of sulfate groups in compound 2. The HMBC correlation between δ _H 7.50 (H-4) protons and δ _C 123.9 (C-5) carbons and between δ _H 7.45 (H-6) protons and δ _C 149.0 (C-8) Indicating that the sulfate is substituted. The ¹ H- ¹ H COZY correlation of H-5 / H-6 and H-6 / H-7 supported the substitution of sulfate at C-8. Thus, compound 2 was identified as 3-hydroxyquinolin-8-yl hydrogen sulfate, named jineol-8-sulfate.

Compound 2: yellow amorphous powder; UV (MeOH)? Max (log?) 242 (4.49), 326 (3.67) nm; IR (KBr)? Max 3385, 1606, 1550, cm ^-1 ; ¹ H and ¹³ C NMR: see Table 2; HR-ESI-MS [M + Na] ⁺ m / z 263.9935 (calculated for C ₉ H ₇ NO ₅ SNa, m / z 263.9943).

Compound 3 was isolated as a yellow amorphous powder. (隆_H 6.35 (d, J = 7.1 Hz; 隆_C 109.7) and 隆_H 7.93 (d, J = 7.1 Hz; 隆_C 140.6) in ¹ H and ¹³ C NMR spectra _The carbonyl carbon in _C 181.0 indicates that compound 3 is a quinolin-4-one alkaloid. The proton signals of δ _C 148.5 at δ _H 7.10 with the quaternary carbon (d, J = 8.0 Hz) , 7.24 (t, J = 8.0 Hz) and _{δ H 7.70 (d, J =} 8.0 Hz) is the compound 3 C Or a quinolone having a hydroxy group at -5 or 8. Compound 3 was determined to be 8-hydroxy-4-quinolone through analysis of HMBC data showing cross peaks between protons of δ _H 7.70 and carbonyl carbon of δ _C 181.0. Compound 3 has a report derived from the ingestion of the moonfish O. dofleini martini (Siuda JF. Et al., Lloydia 1974, 37 (3): 501-503).

Compound 4 was isolated as a colorless solid. NMR spectrum is a typical indole class compound and Compound 4 is coupled via 2- (1 H- indol-3yl) acetic acid via carbonyl carbon at δ _C 176.5 with methylene groups in δ _H 3.96 (s; δ _C 33.5) (Evidente A, et al., Experientia 1993, 49 (2): 182-183) and the structure was further confirmed by analysis of the HMBC spectrum.

Other known alkaloids can be identified by their spectral analysis data comparison and reported in the literature, such as jineol (Moon SS, et al., J Nat Prod 1996,59 (8): 777-779) 3-methoxy-8-hydroxy-quinolin-2-one (Fu YD, et al., J Asian Nat Prod Res 2013, 15 (4): 363-367 4-dimethoxy-8-hydroxy-quinolin-2-one (Yoon MA, et al., Biol Pharm Bull 2006, 29 (4): 735-739), and scolopendrine (Noda N, et al., Chem Pharm Bull 2001, 49 (7): 930-931).

Example 3 Isolation of Human Plasma and Platelets

Human blood samples were taken in the morning from 10 healthy and fast volunteers (age: 24-28 years, 4 males and 6 females) without cardiovascular disease, allergies and fat or carbohydrate metabolic disease, and without medication . All subjects completed their consent before participating in the study. Subjects also did not use antioxidant food supplements and their diet was balanced (meat and vegetables).

Blood was collected from sodium citrate (0.32% final concentration) and immediately centrifuged (2000 rpm x 15 min) to obtain plasma. Human platelets were prepared by the method reported by Fares A et al. (Fares A. et al., N Am J Med Sci 2013, 5 (4): 266-279; Weitz JI, et al., Chest 2004, 126 (3 Suppl): 265S-286S). Briefly, platelet rich plasma (PRP) was prepared by centrifugation at 150 g for 15 minutes at room temperature. PRP was adjusted to a concentration of 1 × 10 ⁹ platelets / mL using a hemocytometer for cell count. PRP is 1 mM CaCl HEPES buffer (5 mM HEPES, 136 mM NaCl , 2.7 mM KCl, 0.42 mM NaH 2 PO4, 2 mM MgCl 2, 5.6 mM glucose, 0.1% BSA (w / v ), pH in the presence of ₂ 7.45). Platelets were left at room temperature for 30 minutes. A 10 milliliter blood sample was used to measure blood coagulation point. The study protocol (KNUH 2012-01-010) was approved by the Institutional Audit Committee of Kyungpook National University Hospital (Daegu, Republic of Korea).

Example 4 Culture of Human Umbilical Vein Endothelial Cells

Primary human umbilical vein endothelial cells (HUVECs) were obtained from Cambrex Bio Science (Charles City, IA, USA) and cultured in a well-known manner (Bae JS, et al., Am J Respir Crit Care Med 2014, 189 (7): 779-786; Ku SK, et al., BMB Rep 2014, 47 (6): 336-341; Ku SK, et al., Arch Pharm Res 2014, 37 (11): 1454-1463; Ku SK, et al., BMB Rep 2014, 47 (7): 376-381). Specifically, cells were cultured in EBM-2 basal medium supplemented with growth supplements (Cambrex Bio Science, Charles City, IA, USA) at 37 ° C under a 5% CO ₂ atmosphere.

<Example 5> Preparation of mice and breeding

Male C57BL / 6 mice (6-7 weeks of age, weighing 27 g) were purchased from Orient Bio Co. (Sungnam, Republic of Korea) and used after a 12-day adaptation period. Mice were housed five per polycarbonate resin under controlled temperature (20-25 ° C) and humidity (40-45% relative humidity) and 12: 12h arm cancer cycles. Mice were fed normal rodent pelleted feeds and arbitrary water during adaptation and processed according to Guidelines for the Management and Use of Laboratory Animals (IRB No. KNU 2012-13) issued by Kyungpook National University, Korea.

<Experimental Example 1> Confirmation of effects of the separated compounds on the coagulation and bleeding time

&Lt; Experimental Example 1-1 > In Vitro ) APTT and PT analysis

The anticoagulant activity of each compound was tested using aPTT and PT assays and human plasma. aPTT and PT were measured according to the manufacturer's instructions using a Thrombotimer (Behnk Elektronik, Norderstedt, Germany). Specifically, transfused normal human plasma (90 μL) was mixed with 10 μL of heparin or each compound and incubated at 37 ° C. for 1 hour. Plasma samples were then incubated for 1 min at 37 ° C after addition of aPTT assay reagent (100 μL) (Fisher Diagnostics, Middletown, VA, USA) and then 20 mM CaCl ₂ (100 μL) . For PT analysis, transfused normal human plasma (90 [mu] L) was mixed with 10 [mu] L of each compound storage solution and incubated at 37 [deg.] C for 1 hour. PT assay reagent (200 μL) (Fisher Diagnostics, Middletown, VA, USA) was pre-incubated at 37 ° C for 10 minutes and then added and the clotting time recorded. PT results were expressed in seconds and expressed as International Standardized Ratio (INR): INR = (PT Sample / PT Control) ^ISI , where ISI = International Sensitivity Index. The aPTT results were only displayed in seconds. For statistical analysis, the results are expressed as mean ± standard deviation of the mean (SEM) of at least three independent experiments performed in duplicate. P <0.05 was considered statistically significant and was determined using SPSS software (version 14.0, SPSS Science, Chicago, IL, USA). Hereinafter, the same method was used for statistical analysis of all experiments.

As a result, cultures of human plasma and Compounds 1 to 4 affected coagulation (Tables 3 and 4).

^a Each value represents the mean ± SEM (n = 5).

^* P < 0.05 compared with control.

^a Each value represents the mean ± SEM (n = 5).

^* P < 0.05 compared with control.

Specifically, compounds 1, 2 and 4 exhibited anticoagulant activity, while compound 3 was weaker than heparin, and aPTT and PT were significantly extended by compounds 1, 2 and 4 (1-5 μM each, More than). However, other compounds such as zineol, 3-methoxy-8-hydroxy-quinoline-2-one, 3,4-dimethoxy-8-hydroxy-quinoline- The coagulation time did not change in the tube. Extension of aPTT and PT suggests the inhibition of a unique or common pathway, and PT extension suggests inhibition of extrinsic or common pathways. Compounds 1, 2 and 4 at concentrations of 3.30, 3.82 and 3.53 μM required twice the coagulation time in the aPTT assay. These results indicated that compounds 1, 2 and 4 can inhibit a common coagulation pathway.

<Experimental Example 1-2> In vitro ( Ex Vivo ) And coagulation and tail bleeding time analysis

In vitro coagulation time and tail bleeding time were measured to confirm in vitro results. Specifically, in vitro coagulation time was determined by fasting male C57BL / 6 mice overnight and then administering each compound contained in 0.5% DMSO as iv. One hour after dosing, arterial blood samples (0.1 mL) were withdrawn in 3.8% sodium citrate (1/10, v / v) for in vitro aPTT and PT measurements. The coagulation time was determined as described above. The tail bleeding time can also be measured using the method described by Dejana et al . ( Thromb Res 1979, 15 (1-2): 191-197) or Kim TH et al . ( J Cell Biochem 2012, 113 (9): 2877-2883) Respectively. Male C57BL / 6 mice were fasted overnight prior to the experiment and were then transversally incised 2 mm from the tip of the mice one hour after intravenous (iv) administration of each compound. Bleeding time was defined as time course until bleeding stopped. Bleeding time over 15 minutes was recorded as lasting for 15 minutes. The mean circulating blood volume for mice is 72 mL / kg (Diehl KH, et al., J Appl Toxicol 2001, 21 (1): 15-23). Since the average weight of the mice used was 27 g, the average blood volume was 2 mL, and the amount of Compound 1 (1.73 or 3.46 μg per mouse), the amount of Compound 2 (1.21 or 2.41 μg per mouse) (0.81 or 1.61 [mu] g per mouse), and the amount of Compound 4 (0.87 or 1.75 [mu] g per mouse) were equal to peripheral blood concentrations of approximately 2.5 or 5.0 [mu] M, respectively.

As a result, as shown in Tables 3 and 4, the tail bleeding time was considerably extended by Compounds 1, 2 and 4 (i.v. injection) as compared to the control. The anticoagulant effect of each compound was shown to be a concentration-dependent prolongation of aPTT and PT (Table 5) as well as in vitro in mice. However, the in vitro blood coagulation time was significantly lower in the other compounds, zineol, 3-methoxy-8-hydroxy-quinoline-2-one, 3,4-dimethoxy-8-hydroxy- It was not changed by scolopendrine.

^a Each value represents the mean ± SEM (n = 5).

^* P < 0.05 compared with control.

Experimental Example 2: Effect of fibrin polymerization, platelet aggregation, and cell survival of isolated compounds

<Experimental Example 2-1> Thrombin-catalyzed fibrin polymerization analysis

The effect of each compound on fibrin polymerization catalyzed by thrombin in human plasma was determined according to the change in absorbance at 360 nm. Specifically, thrombin-catalyzed polymerization was measured every six seconds for 20 minutes by monitoring turbidity at 360 nm using a spectrophotometer (TECAN, Mannedorf, Switzerland) at ambient temperature. Control plasma and incubated plasma with each compound were diluted 3 times in DMSO and then coagulated with thrombin (final concentration: 0.5 U / mL). The maximum polymerization ratio (Vmax,? MOD / min) of each absorption curve was recorded (Nowak P, et al., Thromb Res 2007, 121 (2): 163-174).

As a result, the incubation of human plasma and Compounds 1, 2 and 4 was significantly reduced at the maximum rate of fibrin polymerization (Fig. 3A). To remove the effect of the pH of the sample, all dilutions were made in 50 mM TBS (pH 7.4). We also evaluated the effect of the same volume of DMSO in human plasma and found that it did not affect clotting. Compound 3 did not inhibit fibrin polymerization induced by thrombin (Figure 3A). Leptillase catalyzed polymerization analysis was performed to see if the reduction in fibrin polymerization observed could be due to a direct effect on thrombin, leading to a reduction in fibrin production rather than the polymerization of fibrin formed. Compounds 1, 2, and 4 significantly reduced the polymerization of leptylase catalysis, which excludes potential direct effects on thrombin.

<Experimental Example 2-2> Platelet aggregation analysis

To confirm the anti-coagulant activity of compounds 1, 2 and 4, U46619 (stable thromboxane A2 analogue / flocculent agent) catalytic platelet aggregation assay was performed. Specifically, in vitro platelet aggregation assays were performed according to the method reported by Kim SY, et al., BMB Rep 2011, 44 (2): 140-145; Lee W, et al., Arch Pharm Res 2015, 38 (5): 893-903). The washed human platelets were incubated for 1, 3, 5 or 10 minutes in DMSO with each compound at the indicated concentrations. Followed by stimulation with U46619 (2 [mu] M) in 0.9% saline solution at 37 [deg.] C for 5 minutes. Platelet aggregation was recorded using a coagulation detector (Chronolog, Havertown, PA, USA). In addition, in vitro platelet aggregation assay, male mice were fasted overnight and administered the indicated concentrations of each compound contained in DMSO by intravenous (iv) injection. After 24 hours, platelet-rich plasma (109 platelets / mL) was incubated at 37 ° C for 1.5 minutes in a flocculation detection system under continuous agitation at 1000 rpm in a volume of 240 μL, and then U46619 (final concentration 2 μM) (Calbiochem- Novabiochem Corp., San Diego, Calif., USA). Platelet aggregation was recorded as described above.

As a result, treatment of compounds 1, 2 and 4 significantly inhibited human platelet aggregation induced by U46619 in a concentration-dependent manner (FIG. 3B). The in vitro results were confirmed in an in vitro platelet aggregation assay (i.v. injection, Fig. 3C).

<Experimental Example 2-3> Cell survival analysis

To investigate the effect of the compounds 1, 2 and 4 on cell viability, cell viability analysis was performed by MTT assay. Specifically, MTT assay was used as an indicator of cell viability. Cells 5 × 10 ³ and cultured in 96-well plates at a density of cell / well. After 24 hours, the cells were washed with fresh medium and then treated with each compound. After 48 hours of incubation, the cells were washed and then 100 μL of 1 mg / mL MTT was added to the cells and incubated for 4 hours. Finally, 150 μL of dimethylsulfoxide (DMSO) was added for solubilization of the formed forma salt and then determined by measuring the absorbance at 540 nm using a microplate reader (Tecan Austria GmbH, Grodig, Austria).

As a result, each compound did not affect cell viability to a concentration of 10 [mu] M as determined using MTT assay on HUVECs treated with compound for 24 hours (FIG. 3D).

<Experimental Example 3> In the arterial thrombosis and pulmonary thrombosis models of the separated compounds, In Vivo ) Check the effect

<Experimental Example 3-1> Ferric chloride (FeCl ₃ ) -Induced carotid thrombosis mouse model

Carotid thrombosis mouse models have generally been used to assess antiplatelet effects (Izuhara Y, et al., Arterioscler Thromb Vasc Biol 2008, 28 (4): 672-677). Specifically, a ferric chloride (FeCl ₃ ) induced thrombosis model was established by the method reported by Izuhara Y et al. (Izuhara Y, et al., Arterioscler Thromb Vasc Biol 2008, 28 (4): 672-677). Specifically, male C57BL / 6 mice were fasted overnight and then each labeled compound contained in DMSO was administered by intravenous injection. After anesthesia with 3% isoflurane (Forane, Choongwae Pharma Corp., Seoul, Korea), 0.1 mL of 0.1% rhodamine 6G (Sigma) was intravenously injected. The testis artery (diameter 200 m) was carefully exposed and wetted cotton wool (0.2 mm diameter) with 0.25 mol / L FeCl ₃ was applied to the outer surface of the outer membrane. After 5 minutes, the cotton yarn was removed, and the wound was washed with physiological saline. Thrombus formation was monitored at 35 ° C by three-dimensional imaging with the previously reported method (Goto S, et al., J Am Coll Cardiol 2004, 44 (2): 316-323). After monitoring the size and time of thrombus formation, the results were classified as follows: Thrombocephaly as a bloody thrombus is zero; Small thrombus (50 μm × 75 μm) is 1; A medium-sized thrombus (100 μm × 150 μm) is 2; A large thrombus (200 μm × 300 μm) is 3. The time from the FeCl ₃ -mediated endothelial damage to the occlusion of the testis by large thrombosis was measured. The time of thrombus formation and the size of the thrombus produced are shown in FIG.

As a result, the control mice showed endothelial damage after treatment with FeCl ₃ and resulted in a large thrombus growth at 8.5 ± 0.9 minutes. The antiplatelet GP IIb / IIIa inhibitor tirofiban showed a large hematopoietic growth up to 56.5 ± 5.4 minutes Significantly slowed. Compounds 1, 2, and 4 significantly slowed the formation of thrombus (Figure 4A). 60 minutes after the endothelial damage induced by FeCl ₃ the results of the investigation of the effect of the compounds on the size of blood clots, the compounds 1,2 and 4 reduced the thrombus induced by FeCl ₃ (Fig. 4B).

<Experimental Example 3-2> Preparation and analysis of a mouse model of acute thrombosis induced by combination of collagen and epinephrine

Male C57BL / 6 mice were grouped into groups of 10 after overnight fasting. Each compound suspended in DMSO was intravenously administered to mice. A mixture of epinephrine (50 μg / kg) and collagen (500 μg / kg) was injected into the tail vein of the mice to induce acute thrombosis 1 hour later. Each mouse was carefully observed for 15 minutes to determine if the mice were paralyzed, died, or recovered from acute thrombotic events. For statistical analysis, five separate experiments were performed.

As a result, intravenous injection of a mixture of collagen and epinephrine into a mouse with a large lung thrombosis causing acute paralysis eventually resulted in sudden death (90-95% mortality). The mortality rates of Compounds 1, 2 and 4 treated in each group were significantly reduced compared to the collagen and epinephrine treated groups (FIG. 4C).

Experimental Example 4 Protein kinase C activity and intracellular Ca activity of the isolated compounds ²⁺  Check the effect on movement

The sensitivity of each compound to the signal pathway regulating platelet aggregation was examined. Phospholipase C (PLC) hydrolyzes phosphatidylinositol 4,5-bisphosphate to diacylglycerol and inositol-1,4,5-triphosphate upon platelet stimulation by an agonist, which are each conjugated to protein kinase C (PKC ) And the increase of cytoplasmic Ca ²⁺ . PKC and Ca ²⁺ act synergistically to induce the activity of glycoprotein (GP) IIb / IIIa, a receptor responsible for granule secretion and platelet aggregation (Wei AH, et al., Br J Haematol 2009, 147 (4): 415-430).

< Experimental Example  4-1> Protein Kinase  C activity assay

The effect of each compound on PKC activity was determined by measuring the phosphorylation of MARCKS, a key substrate of PKC in human platelets (Elzagallaai A, et al., Blood 2000, 95 (3): 894-902). Specifically, washed human platelets were incubated with DMSO or each compound (5 [mu] M) for 10 min at 37 [deg.] C and then stimulated with U46619 (2 [mu] M) or thrombin (0.05 U / Phosphorylated-MARCKS in seafood was detected using Western blotting. Western blotting was performed as follows. Whole cell extracts were prepared by dissolving HUVEC and protein concentration was determined using Bradford assay. The same amount of protein was separated by SDS-PAGE (10%) and electroblotted overnight on an emmobilone membrane (Millipore, Billerica, MA, USA). The membrane was washed with 0.05% Tween  (50 mM Tris-HCl, pH 7.5, 150 mM NaCl) containing 5% low fat milk powder for 1 hour. After incubation with phospho-MARCKS (Santa Cruz, CA, USA) for 1.5 hours at room temperature, the cells were incubated with secondary antibody conjugated with mustard radish peroxidase. , et al., BMB Rep 2014, 47 (6): 336-341). β-actin (1: 1000, Santa Cruz, Calif., USA) was used as a loading control.

As a result, at the concentrations required to prevent platelet aggregation, compounds 1, 2 and 4 inhibited MARCKS phosphorylation, respectively, induced by U46619 and thrombin (FIG. 5A).

&Lt; Experimental Example 4-2 > ²⁺  Mobilization analysis

Changes in intracellular [Ca ^{2 +} ] _i were measured by monitoring the fluorescence of fura-2 on platelets loaded with U46619 and thrombin-stimulated fura-2. Specifically, the Ca ^{2 +} mobilization in cells of the platelet ([Ca ^{2 +]} _i) was measured by the method reported by Wu CC (Wu CC, et al ., Planta Med 2007, 73 (5): 439-443). Platelets were incubated with fura-2 / AM (3 [mu] M) for 30 min at 37 [deg.] C. After two washes, platelets loaded with fura-2 were suspended in Ca ^{2 +} -free Tyrode's solution at a final concentration of 5 × 10 ⁷ platelets / mL. Calcium 1 mM was added to platelets loaded with fura-2 1 min before stimulation with platelet activating agent. Fluorescence (Ex 339 nm, Em 500 nm) was measured with a fluorescence photometer (TECAN, Mannedorf, Switzerland). [Ca ^&lt; ^{2 + &} gt ^; ] _i is determined by Grynkiewicz et al. ( J Biol Chem 1985, 260 (6): 3440-3450).

As a result, compounds 1, 2 and 4 inhibited the increase in [Ca ^{2 +} ] _i induced by U46619 and thrombin (FIG. 5B and FIG. 5C).

Experimental Example 5: Effect of isolated compounds on thrombin and FXa activity

In order to illustrate the mechanism for the inhibition of coagulation by compounds 1, 2 and 4, the inhibition of thrombin and FXa activity was measured using a chromogenic substrate.

<Experimental Example 5-1> Analysis of thrombin activity

50 mM Tris-HCl buffer (pH 7.4) containing 7.5 mM EDTA containing each compound and 150 mM NaCl were mixed. After incubation for 2 minutes at 37 ° C, thrombin solution (150 μL, 10 U / mL) (Haematologic Technologies, Essex Junction, VT, USA) was added and incubated for 1 minute at 37 ° C. Then, a solution of S-2238 (thrombin substrate, 150 μL, 1.5 mM) was added and absorbance was monitored for 120 seconds using a spectrophotometer (TECAN, Mannedorf, Switzerland) at 405 nm. A direct thrombin inhibitor, argatroban (Santa Cruz Biotechnology, Inc., Dallas, Tex., USA) was used as a positive control.

 As a result, treatment of Compounds 1, 2 and 4 resulted in a concentration-dependent inhibition of the amidolytic activity of thrombin, indicating direct inhibition of thrombin activity by Compounds 1, 2 and 4 (Fig. 6A).

<Experimental Example 5-2> Analysis of factor Xa activity

FXa analysis was performed in the same manner as described for thrombin activity analysis except that FXa (1 U / mL) and S-2222 (Chromogenix AB, Molndal, Sweden) were used as substrates. A direct FXa inhibitor, rivaroxaban (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) was used as a positive control.

As a result, the compounds 1, 2 and 4 inhibited the activity of FXa (Fig. 6B). This result was in agreement with the results of the antithrombin assay, suggesting that the fundamental antithrombin mechanism of action of compounds 1, 2 and 4 is fibrin polymerization and / or inhibition of the endogenous / exogenous pathway.

Experimental Example 6 Confirmation of Effect of Separated Compounds on Thrombin and FXa Production

Previous studies have shown that endothelial cells can support prothrombin activity by FXa (Sugo T, et al., J Biochem 1995, 117 (2): 244-250). In addition, endothelial cells have been reported to provide functional equivalence of clotogenic phospholipids and support FX activity (Rao LV, et al., Blood 1988, 71 (3): 791-796). In addition, the activity of FX by FVIIa in HUVECs stimulated with TNF-α has been reported to be dependent on TF expression (Ghosh S, et al., J Thromb Haemost 2007, 5 (2): 336-346). Thus, the effects of compounds 1, 2 and 4 were investigated for the activity of FX by FVIIa.

<Experimental Example 6-1> Production of thrombin on the surface of HUVEC

Production of thrombin by HUVEC was quantitated by the method reported by Dejana E et al. (Dejana E, et al., J Cell Biochem 2012, 113 (9): 2877-2883; Bae JS et al., Food Chem Toxicol 2011, 49 (7): 1572-1577). Specifically, HUVECs were preincubated for 10 min in 300 μL of 50 mM Tris-HCl buffer, 100 pM FVa, and 1 nM FXa containing each compound and then incubated with prothrombin (Haematologic Technologies, Essex, Junction, VT, USA). After 10 minutes, a beach sample (10 μL each) was transferred to a 96-well plate containing 40 μL of 0.5 M EDTA-containing TBS per well to terminate prothrombin activity. Activated prothrombin was determined by measuring the rate of hydrolysis of S-2238 (thrombin substrate) (Chromogenix AB, Molndal, Sweden) at 405 nm. Standard curves were prepared with known amounts of purified thrombin.

As a result, the pre-culture with FVa and FXa the HUVEC in the presence of CaCl ₂ before the addition of prothrombin is caused a thrombin production (Fig. 6C). In addition, treatment of compounds 1, 2 and 4 caused concentration-dependent inhibition of thrombin produced by prothrombin (Figure 6C).

<Experimental Example 6-2> Production of factor Xa on the surface of HUVEC

The fusion monolayers of HUVEC (10 ng / mL for 6 hours in serum-free medium) stimulated with TNF-alpha (Abnova, Taipei, Taiwan) in 96-well culture plates (preincubated with indicated concentrations of each compound for 10 minutes ) Was incubated with buffer B (5 mg / mL bovine serum albumin (10 nM)) containing FVIIa (10 nM) for 5 min at 37 ° C in the presence or absence of anti-TF IgG (25 μg / mL) (Santa Cruz, [BSA] and 5 mM CaCl ₂ supplemented buffer A (10 mM HEPES, pH 7.45, 150 mM NaCl, 4 mM KCl, and 11 mM glucose). FX (175 nM) was then added to the cells in a final reaction mixture volume of 100 [mu] L, and then the cells were incubated for 15 minutes. The reaction was stopped by addition of buffer A containing 10 mM EDTA and the amount of FXa produced was measured using a chromogenic substrate. The change in absorbance at 405 nm over 2 minutes was monitored using a microplate reader (Tecan Austria GmbH, Grodig, Austria). The initial color development rate was converted to FXa concentration using a standard curve prepared with a known dilution of purified human FXa.

The result was a 13-fold increase in the rate of FX activity compared to unstimulated HUVEC (7.5 ± 0.5 nM) in HUVEC (98.5 ± 4.5 nM) stimulated with TNF-α to induce TF expression, TF IgG (18.4 +/- 2.2 nM) (Figure 6D). In addition, preincubation with compounds 1, 2 and 4 resulted in a concentration-dependent inhibition of FX activity by FVIIa (Fig. 6D). Therefore, this result suggests that compounds 1, 2 and 4 can inhibit the production of thrombin and FXa.

<Experimental Example 7> Confirmation of effect of isolated compounds on PAI-1 and t-PA secretion

TNF-a is known to inhibit fibrinolysis in HUVEC by inducing production of PAI-I. Changes in the balance between t-PA and PAI-1 are known to cause changes in aggregation and fibrinolysis (Philip-Joet F, et al., Eur Respir J 1995, 8 (8): 1352-1356; Schleef RR , et al., J Biol Chem 1988, 263 (12): 5797-5803). TNF-a has no significant effect on the production of t-PA (Hamaguchi E, et al., J Pharmacol Exp Ther 2003, 307 (3): 987-994), the plasminogen activator and its inhibitors Balance reflects pure plasminogen activity (Davie EW. Et al., Thromb Haemost 1995, 74 (1): 1-6; Davie EW, et al., Biochemistry 1991, 30 (43): 10363-10370 ; Quinn C, et al., Clin Lab Sci 2000, 13 (4): 229-238).

<Experimental Example 7-1> Analysis of PAI-1 secretion

To determine the direct effect of each compound on the TNF-a stimulation secretion of PAI-1, HUVECs were cultured in medium with or without each compound in the absence or presence of TNF-a for 18 hours. The concentration of PAI-1 in the HUVEC culture supernatant was determined using an ELISA kit (American Diagnostica, Inc., Stamford, CT, USA).

As a result, treatment of compounds 1, 2 and 4 resulted in a concentration-dependent inhibition of secretion of TNF-α-induced secretion of PAI-1 from HUVEC, and in each of compounds 1, 2 and 4 (Fig. 7A).

<Experimental Example 7-2> Analysis of t-PA secretion

The effect of TNF-a and compounds 1, 2 and 4 on the secretion of t-PA by HUVEC was investigated. The concentration of t-PA in the HUVEC culture supernatant was determined using an ELISA kit (American Diagnostica, Inc., Stamford, CT, USA). As a result, a decrease in the production of t-PA by TNF-α in HUVEC was consistent with previous reports reported (Lopez S, et al., Atherosclerosis 2000, 152 (2): 359-366). This decrease was not significantly altered by treatment with compounds 1, 2 and 4 (Fig. 7B). These results indicate that TNF- [alpha] increases the PAI-1 / t-PA ratio, which is inhibited by Compounds 1, 2 and 4 (Fig. 7C).

Claims (11)

Characterized by having antithrombotic, anti-platelet or anticoagulant activity due to the prolongation of prothrombin time (PT) and activated partial thromboplastin time (aPTT) of Scolopendra subspinipes mutilans L 0.0 &gt; (Koch) &lt; / RTI &gt;
[Chemical Formula 1]

; And
(2)

.
delete delete delete A prothrombin time (PT) and an activated partial thromboplastin time (PT) containing the compound represented by the following formula (1), (2) or (3) isolated from Scolopendra subspinipes mutilans L. thromboplastin &lt; / RTI &gt; time, aPTT).
[Chemical Formula 1]

;
(2)

; And
(3)

.
6. The composition of claim 5, wherein the composition further comprises prothrombin time (PT) and activated partial thromboplastin time (aPTT), further comprising a pharmaceutical diluent, excipient or carrier. Antithrombotic, or anticoagulant pharmaceutical composition.
A prothrombin time (PT) and an activated partial thromboplastin time (PT) containing the compound represented by the following formula (1), (2) or (3) isolated from Scolopendra subspinipes mutilans L. thromboplastin time, aPTT), an antithrombotic, anti-platelet or anti-hypertensive health functional food:
[Chemical Formula 1]

;
(2)

; And
(3)

.
A prothrombin time (PT) and an activated partial thromboplastin time (PT) containing the compound represented by the following formula (1), (2) or (3) isolated from Scolopendra subspinipes mutilans L. thromboplastin time, aPTT), which is selected from myocardial infarction or ischemic stroke.
[Chemical Formula 1]

;
(2)

; And
(3)

.
delete A prothrombin time (PT) and an activated partial thromboplastin time (PT) containing the compound represented by the following formula (1), (2) or (3) isolated from Scolopendra subspinipes mutilans L. thromboplastin time, aPTT), which is selected from myocardial infarction or ischemic stroke.
[Chemical Formula 1]

;
(2)

; And
(3)

.
delete

Images